Phytochemical Investigation on Euphorbia macrostegia (Persian wood spurge).

Euphorbia macrostegia or Persian wood spurge is one of the seventeen endemic plants of this genus in Iran. Three triterpenoids, 24-methylenecycloartan-3β-ol (1), butyrospermol (2) and cycloartenol (3) and three diglycerides, 1,2-di-O-α-linolenoyl-sn-glycerol (4), 1-O-linoleoyl-3-O-palmitoyl-sn-glycerol (5) and 1-O-α-linolenoyl-2-O-palmitoyl-sn-glycerol (6) were isolated from the hexane soluble part of methanol-dichloromethane extracts of the aerial parts of Euphorbia macrostegia Boiss. The structures of all compounds were elucidated using different spectroscopy methods including, 1H NMR, 13C NMR, HSQC, HMBC, EI-MS and IR. The triterpenes and the unsaturated fatty acids moieties of the diglycerides isolated from the plant were reported previously to have analgesic, anticancer, bactericidal and antifungal activity. Here, we show that E. macrostegia is a new source for the above mentioned biologically active compounds.


Introduction
Euphorbia macrostegia (Persian wood spurge) is one of the seventeen endemic plants of this genus in Iran (1). It grows wild in different parts of Iran including Fars, Kohgiluyeh and Boyer-Ahmad Provinces (2). The plants of the genus Euphorbia are known to have cycloartane triterpenoids, long chain hydrocarbons and fatty acids as their major non-polar constituents (3).

Isolation of compounds from the hexane fraction
The hexane extract was subjected to column chromatography (50 × 5 cm) over silica gel (150 g, 70-230 mesh). The elution of the column was performed using n-hexane with 10% gradient of DCM up to 100%, and then followed by increasing the polarity of the mobile phase with MeOH to afford 48 fractions. Similar fractions were combined based on their similarity in composition, deduced from TLC analyses. Fractions 17-19 (570 mg) were combined and subjected to 5% AgNO 3 -silica gel (60 g) impregnated FCC (30 ×3 cm) (11). The column was eluted with DCM with increasing the polarity to ethyl acetate (EtOAc) that yielded compounds 1 (15 mg) and 2 (45 mg) as a mixture (their ratios were calculated on the basis of their proton᾽s signal integration in the 1 H NMR spectrum) and 3 (20 mg). Fractions 24-27 (170 mg) were pooled and were separated on a FCC (17 g, 20× 2 cm) using DCM with a gradual increasing of the polarity with EtOAc as the mobile phase. Compound 4 (7.2 mg) was purified from the above column. Fractions 28 to 32 (1.4 g) were mixed and loaded on a flash silica gel column (100 g, 50 × 4 cm) and eluted with DCM with a gradient of the polarity to acetone. It afforded 40 fractions. Fraction mixture 17-28 (70 mg) was further purified by repeated FCC to yield compound 5 (9.1 mg) and 6 (16.2 mg).

Spectral data
The structures of all compounds isolated from E. macrostegia were elucidated using spectroscopy methods including 1 H NMR, 13 C NMR, EIMS, IR and by comparison of their spectra with those published in the literature for the authentic samples (5,8,(12)(13)(14)(15)(16).
from dichloromethane extract of E. macrostegia (10) among which 24-methylenecycloartan-3βol was the only common compound with our report. The cytotoxic activities of the isolated compounds on two human cancer cell lines were determined, but the less polar triterpenoids, 24-methylenecycloartan-3β-ol did not show high anticancer activity against the tested cell lines (10).

General
The purified compounds were subjected to NMR measurements on Bruker Avance DRX500 Spectrometer (500 MHz for 1 H and 125 MHz for 13 C). 1 H-and 13 C-NMR were measured in CDCl 3 with TMS as internal standard. Mass spectra (EI-MS) were recorded on an Agilent 5975C inert GC/MSD instrument. IR spectra were recorded on a Perkin Elmer Spectrum One FT-IR spectrometer in CHCl 3 . The chromatography separations were performed using gravity columns chromatography with silica gel 60 (70-230 mesh, 0.063-0.200 mm particle size) and using flash column chromatography (FCC) with silica gel 60 (230-400 mesh, 0.040-0.063mm particle size) and thin layer chromatography (TLC) using silica gel 60 F 254 pre-coated plates (0.25 mm). The above adsorbents were purchased from Merck Chemical Company, Darmstadt, Germany.

Plant material
The aerial parts of E. macrostegia Bioss. in the flowering stage were collected from Chelegah (N 30° 17′, E 51° 56′; at 2370 m altitude ) near Sepidan (Ardakan-e-Fars) Fars, Iran, in July 2012 and the plant was identified by Mojtaba Asadollahi in Medicinal and Natural Product Chemistry Research Center (MNCRC), Shiraz University of Medical Sciences. A voucher specimen (PC-91-4-11) has been deposited at the herbarium of (MNCRC).

Methyl transestrification
For confirming the identification of the acyl moieties of the diacylglycerides, they were transformed to their methyl ester derivatives by reaction with BF 3 in MeOH (17). Briefly 2 mg of the compound was added to 500 μL 20% BF 3 in MeOH in a sealed test tube and then heated on hot water bath (70 °C) for 1 h. To the above solution, 1 mL water was added and the mixture was extracted three times with 3 mL n-hexane. The organic layer were mixed and dried over anhydrous Na 2 SO 4 and evaporated under nitrogen stream. The residue dissolved in 1 mL hexane and then subjected to GC-MS analyses (18).

GC-MS analysis
The GC-MS analytical condition was the same as reported previously (18). The methyl esters of the fatty acids resulting from transesterification of compounds 4-6 were identified by comparison of their retention times (19) and mass spectra recorded on GC-MS with those published in the literature (18,20). The results were in good agreement with those obtained from infra-red (IR), EIMS, 1D and 2D NMR spectral data.
Diglycerides (4-6) were isolated from the more polar fraction obtained from the silica gel column chromatography. Their structures were deduced mainly from examinations of 1D and 2D NMR spectra. In the 1 H NMR and 13 C NMR spectra of different glycerides, the approximate positions of the acyl groups can be deduced from the chemical shifts of the signals of the glycerol protons and carbons (13,14). The priority of the 13 C NMR spectral data is that the length and degree of unsaturation of the acyl chain does not influence the chemical shift of glycerol carbons chemical shifts but they may alter slightly the chemical shift of the respective protons (13,14). The 1 H NMR spectral data of the acyl chains of the glycerides (4-6) were assigned with  comparison to those published in the literature (13,15,16).
For compound 4, the molecular ion was detected at m/z 612 [M] + in EI-MS. APT 13 C NMR spectrum showed three signals at δ 61.1, 67.8, 61.1 ppm for the glycerol᾽s C-1, C-2 and C-3 carbon atoms, respectively that confirm the 1,2-diglyceride structure (13). In the 1 H NMR the signals of the protons of the glycerol moiety appeared at δ 4.3 ppm (2H, dd, J = 4.0, 11.9, H-1b, H-3b), 4.15 (2H, dd, J = 6.0, 11.7, H-1a, H-3a), and the proton signal of H-2 resonated at δ 5.26 ppm which overlapped with the olefinic protons region of the unsaturated acyl moiety and confirmed the substitution of an acyl group at C-2 OH group. The presence of the olefinic protons of the unsaturated acyl chain resonating at δ 5.3-5.4 ppm (m) and six carbon signals at δ 130.9, 129.2, 127.3, 127.2, 126.7, 126.1 ppm suggested three double bonds in the acyl chain. The 1 H NMR and 13 C NMR spectral data of the compound suggested the presence of two linolenyl acyl groups in the molecule.
In the 13 C NMR spectrum of compound 5, two signals for the glycerol carbon atoms appeared at δ 64.0 (C-1, C-3) and 67.3 ppm (C-2) and the respective protons observed at δ 4.07 (2H, dd, J = 5.6, 11.5, H-1a, H-3a), 4.11 (2H, dd, J = 4.2, 11.5, H-1b, H-3b) and 4.05 ppm (1H, m, H-2) in the 1 H NMR spectrum suggested the 1,3-diglyceride substitution (13). On the basis of GC-MS analyses of the transesterified products of the compound, one of the acyl chains was suggested to be linoleic acid and the other was identified as palmitic acid methyl ester. The above finding was compatible with the 1 H NMR spectral data with observing the terminal methyl group of the saturated chain resonated at δ 0.81 ppm while the value was recorded at 0.91 ppm for the linoleyl moiety in addition to four olefinic protons at 5. 2-5.3 ppm (m). In addition to the 1D NMR data, the substitutions at C-1 and C-3 was confirmed by observing the cross peaks between H-1,3; H-2′, H-2″ and C-1′, C-1″ in the HMBC spectrum.
Compound 1 was isolated in several members of the genus Euphorbia and reported to be cytotoxic against p-388 cells and lymphocytic leukemia system at 2.5 μg/mL and more than 20 μg/mL IC 50 s, respectively (4). It is reported as antiproteolytic (21), and causing a dosedependent decrease in lymphocyte proliferation which is suggested to be due to the presence of the free C-3-OH group in the molecule (5). It shows pain-relieving activity and antiinflammatory effect (5).
Compound 2 was purified from "shea-butter" which is prepared from the kernels of the fruit of Butyrospermum parkii, an African medicinal tree (22). It could be used in formulation of pharmaceutical compositions such as in tablets, parenteral solutions and ointments to cure acne, skin cracks, in sun and skin creams (22,23). The compound is reported to have hormonal and bactericidal activity (22).
Cycloartenol (3) and 24-methylenecycloartan-3β-ol (1) are the two major constituent of E. broteri a herbaceous shrub and according to the taxonomic classification suggested by Ponsinet and Ourisson may be in the same clade as E. macrosteigia (8,24).
Different biological activities were reported for linolenic acid and its derivatives such as 1,2-Di-O-α-linolenoyl-3-O-β-galactosyl-snglycerol which was identified as a superoxide generation inhibitor and decrease the O 2 level in the HL-60 assay system. Reactive oxygen and nitrogen species are toxic molecules against pathogens in the immunological defense system (25). The compound was also reported as a feeding stimulant for Plagiodera versiclora a willow beetle living on Salix integra (26).
The anticancer, analgesic, and (3) bactericidal activity of the triterpernoids, 24-methylenecycloartan-3β-ol, butyrospermol and cycloartenol together with antifungal (27) and antibacterial activity (28) of unsaturated fatty acids suggest the Persian Spurge, Euphorbia macrostegia, is a new source for extraction of the biological active natural products. Although the three diglycerides (4-6) have not shown the above-mentioned biological activities, but their acyl chains such as linolenic and linoleic acid are known to have important biological activities like antimicrobial and antifungal activity especially against important plant pathogens such as Rhizoctonia solani, Phythium ultimum, Pyrenophora avenae and Crinipellis perniciosa (27). The above results encourage us to pay more attention to the biological activity of Euphorbia macrostegia in our future investigation.